Throughout this application various publications are referred to by number in square brackets. Full citations for the references may be found at the end of the specification. The disclosures of each of these publications, and also the disclosures of all patents, patent application publications and books recited herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
The disease tuberculosis (TB) remains a global public health problem. An estimated 8.8 million new cases occurred in 2010 with ˜1.1 million TB-associated deaths among HIV− and ˜0.35 million among HIV+ people [12]. Rapid TB diagnosis and treatment leads to reduced transmission, morbidity and mortality. However, diagnosis of TB is often delayed, especially in resource-limited settings where the vast majority of people with TB reside.
The clinical presentations of TB are manifold and diagnostic confirmation can be challenging. The gold standard test for TB diagnosis is detection of Mycobacterium tuberculosis (Mtb) in culture [13]. However, culture methods require laboratory infrastructure and have long turn-around-times of weeks to months. Molecular methods detecting Mtb-specific nucleic acids, especially the recently WHO endorsed GeneXpert MTB/RIF, have revolutionized the rapid diagnosis of drug-sensitive and resistant TB, but are costly and require technological investment [14-17]. Despite ongoing research to identify new biomarkers for TB, a simple cheap point of care (POC) test, applicable in all settings, is still not available [17, 18]. Therefore, although limited by a sensitivity of around 50% in detecting acid fast bacilli (AFB) in sputum smears [19-21], microscopy remains the most widely used method for rapid TB diagnosis, and often is the only test available in resource-limited settings.
Nevertheless, even microscopy requires collection and processing of at least 2 sputum samples, and it can take up to 2 days until results are available. Furthermore, these modalities require either sputum or another specimen from the site of disease, which may not be available. A novel urine antigen detection test for TB appears to have clinical value in patients with advanced HIV infection but not in HIV negative (HIV−) patients, which constitute a large portion of TB cases [22-25].
To complement the current armamentarium of diagnostic tests, TB biomarkers should be detectable in an easily accessible sample independent of the site of disease (e.g. blood or urine) and have the potential to be adapted into a rapid test requiring minimal to no laboratory infrastructure.
As another approach, the amplifying power of the immune responses can potentially detect infection with Mtb at a low antigen-threshold and distant from the site of infection. Assays that detect Mtb infection by measuring interferon gamma release of circulating lymphocytes in response to Mtb-specific antigens (IGRAs) are more accurate than the tuberculin skin test (TST) [26]. However, they require incubation of whole blood cells, which is not feasible in most resource-limited settings. More importantly, neither the TST nor IGRAs can distinguish TB from the asymptomatic state of infection, latent TB infection (LTBI) (reviewed in [27]). In contrast, assays detecting serum antibodies (Abs) can be scaled up into rapid, robust, inexpensive and simple formats requiring little laboratory infrastructure. Furthermore, it has been demonstrated that Ab responses to mycobacterial antigens can discriminate between TB and LTBI (reviewed in [28] and [29, 30]). However, commercially available serodiagnostic tests thus far are limited by insufficient sensitivity and specificity (reviewed in [5-7]). Given their poor performance, the WHO recently recommended against the use of commercial serodiagnostic tests, while encouraging further targeted research in this field [31].
The present invention addresses the need for improved rapid and simple tuberculosis disease assays.